Methods for inhibiting deacetylase activity

ABSTRACT

A method for identifying a compound that inhibits the NAD + -dependent deacetylase activity of a SIR2 protein is disclosed. These compounds are useful for the treatment of cancers and other diseases, through the activation of silenced genes, through the promotion of apoptosis in cancerous cells, and through the inhibition of transcriptional repressor activity in oncogenes.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/333,884, filed Nov. 27, 2001. The foregoing application is herebyincorporated by reference in its entirety for all purposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK.

NOT APPLICABLE

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

A portion of the present invention was made under federally sponsoredresearch and development under National Heart, Lung, and Blood InstituteGrant HL04211, National Institutes of Health Grant GM43893, and NationalCancer Institute Grant CA78746. The Government may have rights incertain aspects of this invention.

BACKGROUND OF THE INVENTION

Portions of the eukaryotic genome can be maintained in atranscriptionally inactive, or silenced, state as the result of thelocal chromatin structure. Silent chromatin may encompass regionsranging from a few thousand base pairs, as in the silent mating typegenes of the yeast S. cerevisiae (Loo, S. & Rine, J. (1995) Annu. Rev.Cell Dev. Biol. 11, 519-48), to whole chromosomes, such as the inactiveX-chromosome in mammals (Lyon, M. F. (1999) Curr. Biol. 9, R235-7). Theformation of silent chromatin, which is best understood at the S.cerevisiae silent mating type loci HMR and HML, and telomeres, dependson DNA elements, or silencers. The HM silencers are located in proximityto the genes they regulate and contain a combination of binding sitesfor Raplp, Abflp and the origin recognition complex (ORC) (Loo, S. &Rine, J. (1995) Annu. Rev. Cell Dev. Biol. 11, 519-48). These proteinsrecruit the SIR (Silent Information Regulator) protein complex(Sir2p-4p) through protein-protein interactions. Once recruited tosilencers, the SIR complex is thought to spread along the chromatinthrough binding of Sir3p and Sir4p to the NH₂-terminal tails of histoneH3 and H4 (reviewed in Gartenberg, M. R. (2000) Curr. Opin. Microbiol.3, 132-7). Among the many requirements for silent chromatin (reviewed inWu, J. & Grunstein, M. (2000) Trends Biochem. Sci. 25, 619-23),post-translational modification (i.e. acetylation, phosphorylation,methylation and ubiquitination) of the NH₂-terminal tails of histonesappears to be critical. For example, the tails of histones H3 and H4 arehypoacetylated in silent chromatin compared to other regions of thegenome (Braunstein, M. et al. (1993) Genes Dev. 7, 592-604). Of the SIRproteins, Sir2p has been shown to be an NAD⁺-dependent histonedeacetylase, and is responsible for the hypoacetylated state of histonesin silent chromatin (Moazed, D. (2001) Curr. Opin. Cell Biol. 13, 232-8;Imai, S. et al. (2000) Nature 403, 795-800; Smith, J. S. et al. (2000)Proc. Natl. Acad. Sci. USA 97, 6658-63; Landry, J. et al. (2000) Proc.Natl. Acad. Sci. USA 97, 5807-11). Sir2p also acts at the ribosomal RNAgene cluster (rDNA) in the RENT protein complex, which does not includeSir3p or Sir4p (Straight, A. F. et al. (1999) Cell 97, 245-56), where itacts to repress recombination.

The yeast SIR2 gene is the defining member of a broadly conserved familyof NAD⁺-dependent deacetylases, termed sirtuins, found in organismsranging from bacteria to humans (Frye, R. A. (2000) Biochem. Biophys.Res. Commun. 273, 793-8). Sirtuins are highly conserved and contain aconserved catalytic domain of approximately 275 amino acids (Grozinger,C. M. et al., (2001) J. Biol. Chem. 276, 38837-38843). In S. cerevisiaealone, four additional homologues have been identified, while in humans,eight homologues have been identified (Grozinger, C. M. et al. (2001)supra). The yeast SIR2 gene shares the greatest similarity with genesfound in other eukaryotes, where it is believed that these closelyrelated homologues serve a comparable role in silencing. Interestingly,SIR2 and its homologues have been implicated in the genetic regulationof aging, both in yeast and C. elegans (Tissenbaum, H. A. & Guarente, L.(2001) Nature 410, 227-30; Sinclair, D. A. & Guarente, L. (1997) Cell91, 1033-42) and in metazoan development though the details of how itaffects these fundamental processes are still mysterious.

Recently, several groups (Luo, J. et al. (2001) Cell 107, 137-48; andVaziri, H. et al. (2001) Cell 107, 149-59) have explored the influenceof the mammalian homologues, Sir2α (the mouse homologue of S. cerevisiaeSIR2, also known as mSIRT1) and SIR2α (the human homologue of S.cerevisiae SIR2, also known as hSIRT1), on the activity of the p53 tumorsuppressor gene. These studies indicate that deacetylase activity ofSir2α and SIR2α act on p53, resulting in suppression of the tumorsuppressor activity. They have also shown that this deacetylase activityis dependent on nicotinamide adenosine dinucleotide (NAD).

What is needed in the art, is a method for inhibiting the NAD⁺-dependentdeacetylase activity of a member of the SIR2 family of proteins using asmall molecule. Surprisingly, the present invention meets this need.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for identifyingcompounds useful for the treatment of cancer or genetic blood diseases,comprising the step of determining whether the compound inhibits thedeacetylase activity of a NAD⁺-dependent deacetylase. In a relatedaspect of the present invention, the method for treating cancer orgenetic blood diseases comprises the step of administering to a subjectin need thereof, a therapeutically effective amount of a compound thatinhibits the deacetylase activity of a NAD⁺-dependent deacetylase.

In a second aspect of the present invention, a method is provided foridentifying compounds which will be useful for the treatment of canceror genetic blood diseases, comprising the step of determining whetherthe compound inhibits the NAD⁺-dependent deacetylase activity of amember of the SIR2 family of proteins. In a related aspect of thepresent invention, the method for treating cancer or genetic blooddiseases comprises the step of administering to a subject in thereof, atherapeutically effective amount of a compound that inhibits theNAD⁺-dependent deacetylase activity of a member of the SIR2 family ofproteins.

In a third aspect of the present invention, a method is provided foractivating a silenced gene in a cell, comprising contacting the cellwith an effective amount of a compound which is capable of inhibitingthe NAD⁺-dependent deacetylase activity of a member of the SIR2 familyof proteins.

In a fourth aspect of the present invention, a method is provided forpromoting p53-dependent apoptosis of a cell comprising contacting thecell with an effective amount of a compound which is capable ofinhibiting the NAD⁺-dependent deacetylase activity of a member of theSIR2 family of proteins.

In a further aspect of the present invention, a method is provided forinhibiting BCL6 transcriptional repressor activity, comprisingcontacting a cell with an effective amount of a compound which iscapable of inhibiting the NAD⁺-dependent deacetylase activity of amember of the SIR2 family of proteins.

In another aspect of the present invention, a method is provided forinhibiting the deacetylase activity of a NAD⁺-dependent deacetylasecomprising contacting the NAD⁺-dependent deacetylase with aNAD⁺-dependent deacetylase inhibiting amount of a compound of Formula I:

In Formula I, the letter X is a member selected from the groupconsisting of O and S. The symbols L¹ and L² each represent membersindependently selected from the group consisting of O, S, ethylene andpropylene, substituted with 0-2 R groups, wherein exactly one of thesymbols L¹ and L² represents a member selected from the group consistingof O and S. Each instance of the letter R of symbols L¹ and L²independently represents a member selected from the group consisting ofC₁₋₆-alkyl, C₂₋₆alkenyl and —CO₂R⁴. The symbols R¹ and R² each representmembers independently selected from the group consisting of hydrogen,C₁₋₆alkoxy, C₀₋₆alkoxy-aryl and hydroxy. Alternatively, the symbols R¹and R² are taken together with the carbons to which they are attached toform a six-membered lactone ring. The symbol R³ represents a memberselected from the group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR⁴,—NR⁴R⁴, —CO₂R⁴, —C(O)R⁴, —C(O)NR⁴R⁴, —CN, —NO₂ and halogen. Eachinstance of the symbol R⁴ independently represents a member selectedfrom the group consisting of hydrogen and C₁₋₆alkyl.

In a further aspect of the present invention, a method is provided forinhibiting the deacetylase activity of a NAD⁺-dependent deacetylasecomprising contacting the NAD⁺-dependent deacetylase with aNAD⁺-dependent deacetylase inhibiting amount of a compound of FormulaII:

In Formula II, the symbol R^(a) is a member selected from the groupconsisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e), —NR^(e)R^(e),—CO₂R^(e), —C(O)R^(e), —C(O)NR^(e)R^(e), —CN, —NO₂ and halogen, whilethe symbol R^(b) is a member selected from the group consisting of:

In the components above, the symbol X^(a) represents a member selectedfrom the group consisting of O, S and NR^(e), while the symbol R^(c)represents a member selected from the group consisting of hydrogen,C₁₋₆alkyl and aryl optionally substituted with a member selected fromthe group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e),—NR^(e)R^(e), —CN, —NO₂ and halogen. The symbol R^(d) represents amember selected from the group consisting of hydrogen, C₁₋₆alkyl, aryl,—OR^(e), —NR^(e)R^(e) and halogen. And, each instance of the symbolR^(e) independently represents a member selected from the groupconsisting of hydrogen and C₁₋₆alkyl.

In a further aspect of the present invention, a method is provided forthe treatment of cancer comprising the step of administering to asubject in need of such treatment a first amount of an antineoplasticagent and a second amount of a compound of Formula I:

In Formula I, the letter X is a member selected from the groupconsisting of O and S. The symbols L¹ and L² each represent membersindependently selected from the group consisting of O, S, ethylene andpropylene, substituted with 0-2 R groups, wherein exactly one of thesymbols L¹ and L² represents a member selected from the group consistingof O and S. Each instance of the letter R of symbols L¹ and L²independently represents a member selected from the group consisting ofC₁₋₆alkyl, C₂₋₆alkenyl and —CO₂R⁴. The symbols R¹ and R² each representmembers independently selected from the group consisting of hydrogen,C₁₋₆alkoxy, C₀₋₆alkoxy-aryl and hydroxy. Alternatively, the symbols R¹and R² are taken together with the carbons to which they are attached toform a six-membered lactone ring. The symbol R³ represents a memberselected from the group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR⁴,—NR⁴R⁴, —CO₂R⁴, —C(O)R⁴, —C(O)NR⁴R⁴, —CN, —NO₂ and halogen. Eachinstance of the symbol R⁴ independently represents a member selectedfrom the group consisting of hydrogen and C₁₋₆alkyl.

In another aspect of the present invention, a method is provided for thetreatment of cancer comprising the step of administering to a subject inneed of such treatment a first amount of a an antineoplastic agent, anda second amount of a compound of Formula II:

In Formula II, the symbol R^(a) is a member selected from the groupconsisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e), —NR^(e)R^(e),—CO₂R^(e), —C(O)R^(e), —C(O)NR^(e)R^(e), —CN, —NO₂ and halogen, whilethe symbol R^(b) is a member selected from the group consisting of:

In the components above, the symbol X^(a) represents a member selectedfrom the group consisting of O, S and NR^(e), while the symbol R^(c)represents a member selected from the group consisting of hydrogen,C₁₋₆alkyl and aryl optionally substituted with a member selected fromthe group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e),—NR^(e)R^(e), —CN, —NO₂ and halogen. The symbol R^(d) represents amember selected from the group consisting of hydrogen, C₁₋₆alkyl, aryl,—OR^(e), —NR^(e)R^(e) and halogen. And, each instance of the symbolR^(e) independently represents a member selected from the groupconsisting of hydrogen and C₁₋₆alyl.

In yet another aspect of the present invention, a composition isprovided for the treatment of cancer comprising an antineoplastic agentand a compound of Formula I:

In Formula I, the letter X is a member selected from the groupconsisting of O and S. The symbols L¹ and L² each represent membersindependently selected from the group consisting of O, S, ethylene andpropylene, substituted with 0-2 R groups, wherein exactly one of thesymbols L¹ and L² represents a member selected from the group consistingof O and S. Each instance of the letter R of symbols L¹ and L²independently represents a member selected from the group consisting ofC₁₋₆alkyl, C₂₋₆alkenyl and —CO₂R⁴. The symbols R¹ and R² each representmembers independently selected from the group consisting of hydrogen,C₁₋₆alkoxy, C₀₋₆alkoxy-aryl and hydroxy. Alternatively, the symbols R¹and R² are taken together with the carbons to which they are attached toform a six-membered lactone ring. The symbol R³ represents a memberselected from the group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR⁴,—NR⁴R⁴, —CO₂R⁴, —C(O)R⁴, —C(O)NR⁴R⁴, —CN, —NO₂ and halogen. Eachinstance of the symbol R⁴ independently represents a member selectedfrom the group consisting of hydrogen and C₁₋₆alkyl.

In a further aspect of the present invention, a composition is providedfor the treatment of cancer comprising an antineoplastic agent and acompound of Formula II:

In Formula II, the symbol R^(a) is a member selected from the groupconsisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e), —NR^(e)R^(e),—CO₂R^(e), —C(O)R^(e), —C(O)NR^(e)R^(e), —CN, —NO₂ and halogen, whilethe symbol R^(b) is a member selected from the group consisting of:

In the components above, the symbol X^(a) represents a member selectedfrom the group consisting of O, S and NR^(e), while the symbol R^(c)represents a member selected from the group consisting of hydrogen,C₁₋₆alkyl and aryl optionally substituted with a member selected fromthe group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e),—NR^(e)R^(e), —CN, —NO₂ and halogen. The symbol R^(d) represents amember selected from the group consisting of hydrogen, C₁₋₆alkyl, aryl,—OR^(e), —NR^(e)R^(e) and halogen. And, each instance of the symbolR^(e) independently represents a member selected from the groupconsisting of hydrogen and C₁₋₆alkyl.

In another aspect of the present invention, a pharmaceutical compositionis provided, comprising a pharmaceutically acceptable excipient and acompound, and all pharmaceutically acceptable salts thereof, of FormulaI:

In Formula I, the letter X is a member selected from the groupconsisting of O and S. The symbols L¹ and L² each represent membersindependently selected from the group consisting of O, S, ethylene andpropylene, substituted with 0-2 R groups, wherein exactly one of thesymbols L¹ and L² represents a member selected from the group consistingof O and S. Each instance of the letter R of symbols L¹ and L²independently represents a member selected from the group consisting ofC₁₋₆alkyl, C₂₋₆alkenyl and —CO₂R⁴. The symbols R¹ and R² each representmembers independently selected from the group consisting of hydrogen,C₁₋₆alkoxy, C₀₋₆alkoxy-aryl and hydroxy. Alternatively, the symbols R¹and R² are taken together with the carbons to which they are attached toform a six-membered lactone ring. The symbol R³ represents a memberselected from the group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR⁴,—NR⁴R⁴, —CO₂R⁴, —C(O)R⁴, —C(O)NR⁴R⁴, —CN, —NO₂ and halogen. Eachinstance of the symbol R⁴ independently represents a member selectedfrom the group consisting of hydrogen and C₁₋₆alkyl.

In another aspect of the present invention, a pharmaceutical compositionis provided, comprising a pharmaceutically acceptable excipient and acompound, and all pharmaceutically acceptable salts thereof, of FormulaII:

In Formula II, the symbol R^(a) is a member selected from the groupconsisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e), —NR^(e)R^(e),—CO₂R^(e), —C(O)R^(e), —C(O)NR^(e)R^(e), —CN, —NO₂ and halogen, whilethe symbol R^(b) is a member selected from the group consisting of:

In the components above, the symbol X^(a) represents a member selectedfrom the group consisting of O, S and NR^(e), while the symbol R^(c)represents a member selected from the group consisting of hydrogen,C₁₋₆alkyl and aryl optionally substituted with a member selected fromthe group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e),—NR^(e)R^(e), —CN, —NO₂ and halogen. The symbol R^(d) represents amember selected from the group consisting of hydrogen, C₁₋₆alkyl, aryl,—OR^(e), —NR^(e)R^(e) and halogen. And, each instance of the symbolR^(e) independently represents a member selected from the groupconsisting of hydrogen and C₁₋₆alkyl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A) Chemical structure of S1. (B) Activation of a TRP1 reporterat the silent HMR mating locus by S1 (S). Wild type (SIR2) or sir2Δcells with TRP1 integrated into HMR. Cells were replica plated ontocomplete synthetic media, or media lacking tryptophan (-trp) without orwith the indicated concentrations of S1. (C) Loss of responsiveness to αfactor in the presence of S1. The halo of cells indicates those able togrow. (D) S1 increases recombination of an ADE2 reporter integratedwithin ribosomal DNA array.

FIG. 2. S1-treated wild type (wt) cells and sir2Δ cells display similartranscriptional changes relative to untreated wt cells. (A) Correlationof transcriptional changes between genetic and chemical inactivation ofSir2p. The correlation plot shows transcriptional changes in a sir2Δmutant relative to wt (sir2Δ/wt) on the vertical axis and changes in wtcells treated with S1 relative to untreated wt cells (15 μM S1/notreatment) on the horizontal axis. (B) A Venn diagram comparing genesup-regulated (LEFT) and down-regulated (RIGHT) more than 2-fold relativeto wt or untreated cells and sir2Δ, hst1Δ or S1-treated wt cells. (C)Correlation of transcriptional changes in wt cells in response to S1treatment with and without cycloheximide. The correlation plot showstranscriptional changes in a S1 and cycloheximide-treated wt cellsrelative to cells treated with cycloheximide alone (15 μM S1 CYH/CYH) onthe vertical axis and changes in wt cells treated with S1 relative tountreated wt cells (15 μM S1/no treatment) on the horizontal axis. (D)Venn diagrams comparing transcriptional changes (up- or down-regulation)in hst2Δ, hst3Δ, hst4Δ cells and S1-treated cells (split).

FIG. 3. (A) Inhibition of NAD-dependent histone deacetylase activity(HDA) of Sir2p by S1. (B) Imnmunoblot of Sir2p in whole cell lysatescontaining overexpressed wild type or drug resistant mutant SIR2. (C)Telomeric silencing in SIR2, sir2Δ and drug-resistant SIR2 mutants. (D)Sequence alignment between yeast Sir2p and Hst1-4p. The region displayedin the alignment contains the putative substrate-binding site. Arrowsindicate the positions of residues that, when mutated in Sir2p, conferS1 resistance.

FIG. 4. (A) Cell cycle analysis of a factor arrested MATa cells treatedwith S1. (B) α2 mRNA expression from the silent HML locus in G1-arrestedcells treated with S1. The RNA from MATα and MATa sir2Δ cells isincluded for comparison. The weak lower molecular weight band is due tocross hybridization to a2 mRNA.

FIG. 5. S1 sensitizes mammalian cells to DNA damaging agents. The bargraph inset shows viability of cells treated with S1 relative to vehicletreated control.

FIG. 6. Table of exemplary compounds of the present invention, and theirpotency for the inhibition of the NAD⁺-dependent deacetylase activity ofa member of the SIR2 family of proteins.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures for organicand analytical chemistry are those well known and commonly employed inthe art.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group isone having one or more double bonds or triple bonds. Examples ofunsaturated alkyl groups include vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. The term “alkyl,” unless otherwise noted, is also meant toinclude those derivatives of alkyl defined in more detail below as“heteroalkyl.” Preferred alkyl groups are limited to hydrocarbon groups,and may be branched- or straight-chain. More preferred alkyl groups areunsubstituted.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified by—CH₂CH₂CH₂CH₂—, and further includes those groups described below as“heteroalkylene.” Typically, an alkyl (or alkylene) group will have from1 to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred in the present invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, Si and S, and wherein the nitrogenand sulfur atoms may optionally be oxidized and the nitrogen heteroatommay optionally be quatemized. The heteroatom(s) O, N and S may be placedat any interior position of the heteroalkyl group. The heteroatom Si maybe placed at any position of the heteroalkyl group, including theposition at which the alkyl group is attached to the remainder of themolecule. Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified by—CH₂—CH₂—S—CH₂CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon substituent which can be a single ringor multiple rings (up to three rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quatemized. A heteroaryl group can be attached tothe remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazol 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) are meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —CN and —NO₂ in a number ranging from zero to (2 m′+1),where m is the total number of carbon atoms in such radical. R′, R″ andR′″ each independently refer to hydrogen, unsubstituted (C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens,unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C₁-C₄)alkylgroups. When R′ and R″ are attached to the same nitrogen atom, they canbe combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like). Preferably, substitutedalkyl groups are those having 3, 2 or 1 substituents selected from thegroup consisting of —OR′, —NR′R″, -halogen, —C(O)R′, —CO₂R′, —CONR′R″,—CN and —NO₂.

Similarly, substituents for the aryl and heteroaryl groups are variedand are selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN,—NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′,—NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —N₃, —CH(Ph)₂, perfluoro(C₁-C₄)alkoxy, andperfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total numberof open valences on the aromatic ring system; and where R′, R″ and R′″are independently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. Preferably, substituted arylgroups are those having 1, 2 or 3 substituents selected from the groupconsisting of -halogen, —OR′, —NR′R″, —CN, —NO₂, —CO₂R′, —CONR′R″,—C(O)R′, —N₃, perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula-A-(CH₂)_(r)-B-, wherein A and B are independently —CH₂—, —O—, —NH—,—S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integerof from 1 to 3. One of the single bonds of the new ring so formed mayoptionally be replaced with a double bond. Alternatively, two of thesubstituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituent R′ in —NR′— and —S(O)₂NR′—, is selected from hydrogen orunsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N) and sulfur (S).

As used herein, the term “lactone ring” refers to a five-, six- orseven-membered cyclic ester, such as

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic,citric, tartaric, methanesulfonic, and the like. Also included are saltsof amino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge, S. M., et al, “Pharmaceutical Salts”, Journal of PharmaceuticalScience, 1977, 66, 1-19). Certain specific compounds of the presentinvention contain both basic and acidic functionalities that allow thecompounds to be converted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

The term “SIR2” refers to the silent information regulator family ofproteins, also known as sirtuins. This family includes both mammalianand non-mammalian proteins. For example, yeast homologues of SIR2include, but are not limited to, HST1, HST2, HST3 and HST4. Themammalian homologues include, but are not limited to, SIRT1, SIRT2,SIRT3, SIRT4, SIRT5, SIRT6, SIRT7 and SIRT8, as well as sirtuins 1 to 8.More specific examples include, but are not limited to, Sir2p and SIR2α.

The term “NAD⁺-dependent deacetylase” refers to a protein that removesthe acetyl groups from a lysine residue of another protein, wherein thedeacetylation is coupled to NAD (nicotinamide adenosine dinucleotide)cleavage.

The term “p53-dependent apoptosis” refers to the genetically determineddeath of a cell that is dependent on, or stimulated by, the p53 gene, agene that typically inhibits non-natural cell growth, such as thatobserved in tumors.

The term “BCL6 transcriptional repressor activity” refers to theactivity of the BCL6 gene that results in the repression oftranscription, the process of constructing an RNA chain from a DNAtemplate.

The terms “silence”, “silencing” and “silenced” refers to a mechanism bywhich gene expression in particular regions of the genome are repressed.

The term “chromatin” refers to a complex mixture of nucleic acid andproteins (such as histone) in eukaryotic cells, and is usually dispersedin the interphase nucleus and condensed into chromosomes.

The term “gene” refers to any segment of DNA associated with abiological function. Thus, genes include coding sequences and/or theregulatory sequences required for their expression. Genes also includenonexpressed DNA segments that, for example, form recognition sequencesfor other proteins.

The term “protein” refers to series of amino acid residues connected oneto the other by peptide bonds between the alpha-amino and carboxy groupsof adjacent residues. In general, the term “protein” is used todesignate a series of greater than 50 amino acid residues connected oneto the other.

The term “antineoplastic agent” refers to a means for inhibiting orcombating the undesirable growth of biological tissue. Antineoplasticagents include, but are not limited to, antiangiogenic and antivascularagents, antimetabolites, antifolates and other inhibitors of DNAsynthesis, antisense oligonucleotides, biological response modifiers,DNA-alkylating agents, DNA intercalators, DNA repair agents, growthfactor receptor kinase inhibitors, hormone agents, immunoconjugates,microtubule disruptors and topoisomerase I/II inhibitors. Antineoplasticagents can also include cyclophosphamide, triethylenephosphoramide,triethylenethiophosphoramide, flutamide, altretamine,triethylenemelamine, trimethylolmelamine, meturedepa, uredepa,aminoglutethimide, L-asparaginase, BCNU, benzodepa, bleomycin, busulfan,camptothecin, capecitabine, carboquone, chlorambucil, cytarabine,dactinomycin, daunomycin, daunorubicin, docetaxol, doxorubicin,epirubicin, estramustine, dacarbazine, etoposide, fluorouracil,gemcitabine, hydroxyurea, ifosfamide, improsulfan, mercaptopurine,methotrexate, mitomycin, mitotane, mitoxantrone, novembrichin,paclitaxel, piposulfan, plicamycin, prednimustine, procarbazine,tamoxifen, temozolomide, teniposide, thioguanine, thiotepa, UFT, uracilmustard, vinblastine, vincristine, vinorelbine and vindesine.

The term “cancer” refers to the uncontrolled growth of abnormal cells.Specific cancers are selected from, but not limited to,rhabdomyosarcomas, chorio carcinomas, glioblastoma multiformas (braintumors), bowel and gastric carcinomas, leukemias, ovarian cancers,prostate cancers, lymphomas, osteosarcomas or cancers which havemetastasized.

The term “genetic blood disease” refers to a hereditary disease of theblood that includes, but is not limited to, hyperproliferative diseases,thalassaemias and sickle cell disease.

The term “tumor suppressor gene” refers to a gene that acts to suppressthe uncontrolled growth of a cancer, such as a tumor.

The term “ligand binding domain” refers to a region of a protein,enzyme, or gene that binds to a ligand selective for that particularsite;

The terms “treating” and “treatment” refer to a method of alleviating orabating a disease and/or its attendant symptoms.

The terms “inhibition”, “inhibits” and “inhibitor” refer to a method ofprohibiting a specific action or function.

The term “therapeutically effective amount” refers to that amount of thecompound being administered sufficient to prevent development of oralleviate to some extent one or more of the symptoms of the condition ordisorder being treated.

The term “composition” as used herein is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts. By“pharmaceutically acceptable” it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and deleterious to the recipient thereof.

The term “subject” refers to animals such as mammals, including, but notlimited to, primates (e.g., humans), cows, sheep, goats, horses, dogs,cats, rabbits, rats, mice and the like. In certain embodiments, thesubject is a human.

General

The present invention involves a phenotypic screen for small moleculeinhibitors of the NAD⁺-dependent deacetylase activity of the SIR2 classof proteins. Several of the proteins in this class play an importantrole in the silencing of genes. In one aspect, the deacetylation ofhistone by a protein in the SIR2 class, can lead to the silencing oftumor suppressor genes. In another aspect, the deacetylation of the p53tumor suppressor gene by a protein in the SIR2 class, reducesp53-dependent apoptosis. Diseases in which apoptosis is involved includediseases that are associated with an increase in cell survival due toinhibition of apoptosis, such as cancer, autoimmune diseases,inflammatory diseases and viral infections and diseases that areassociated with a decrease in cell death due to hyperactive apoptosis,such as AIDS, neurodegenerative disease, hematologic diseases, andtissue damage. A further aspect of the present invention relates to theacetylation of BCL6 by inhibiting the deacetylase activity of a proteinin the SIR2 class. Doing so prevents expression of differentiation genesin B-cell non-Hodgkin lymphoma (B-NHL) and diffused large B-celllymphomas (DLBCL). Therefore, inhibiting the NAD⁺-dependent deacetylaseactivity of a protein in the SIR2 family of proteins leads to theactivation of p53 and either growth or arrest of apoptosis, it ispossible to treat various cancers and disease states that are well-knownto one of skill in the art.

EMBODIMENTS OF THE INVENTION

Methods

In view of the surprising discovery above, the present inventionprovides in one aspect a method is provided for identifying compoundsuseful for the treatment of cancer or genetic blood diseases, comprisingthe step of determining whether the compound inhibits the deacetylaseactivity of a NAD⁺-dependent deacetylase. In a related aspect of thepresent invention, the method for treating cancer or genetic blooddiseases comprises the step of administering to a subject in needthereof, a therapeutically effective amount of a compound that inhibitsthe deacetylase activity of a NAD⁺-dependent deacetylase.

In a preferred aspect of the present invention, the identified compoundsare useful for the treatment of silenced tumor suppressor genes,B-cell-derived non-Hodgkin lymphomas and diffuse large B-cell lymphomas.In another preferred aspect of the present invention, the identifiedcompounds are useful for the treatment of thalassaemias and sickle celldisease.

In a further preferred aspect of the present invention, the step ofdetermining comprises the step of specifically binding radiolabelled(1,2-dihydro-3H-naphtho[2,1-b]pyran-3-one) to the ligand binding domainof a member of the SIR2 family of proteins.

In another preferred aspect of the present invention, the NAD⁺-dependentdeacetylase is a member of the SIR2 family of proteins. In a morepreferred aspect, the member of the SIR2 family of proteins is selectedfrom the group consisting of Sir2p and SIR2α. In a most preferredaspect, the member of the SIR2 family of proteins is SIR2α.

In another aspect of the present invention, a method is provided foridentifying compounds which will be useful for the treatment of canceror genetic blood diseases, comprising the step of determining whetherthe compound inhibits the NAD⁺-dependent deacetylase activity of amember of the SIR2 family of proteins. In a preferred aspect of thepresent invention, the method for treating cancer or genetic blooddiseases comprises the step of administering to a subject in thereof, atherapeutically effective amount of a compound that inhibits theNAD⁺-dependent deacetylase activity of a member of the SIR2 family ofproteins.

In another preferred aspect of the present invention, a method isprovided for activating a silenced gene in a cell, comprising contactingthe cell with an effective amount of a compound which is capable ofinhibiting the NAD⁺-dependent deacetylase activity of a member of theSIR2 family of proteins.

In still another preferred aspect of the present invention, a method isprovided for promoting p53-dependent apoptosis of a cell comprisingcontacting the cell with an effective amount of a compound which iscapable of inhibiting the NAD⁺-dependent deacetylase activity of amember of the SIR2 family of proteins.

In a further aspect of the present invention, a method is provided forinhibiting BCL6 transcriptional repressor activity, comprisingcontacting a cell with an effective amount of a compound which iscapable of inhibiting the NAD⁺-dependent deacetylase activity of amember of the SIR2 family of proteins.

In another aspect of the present invention, a method is provided forinhibiting the deacetylase activity of a NAD⁺-dependent deacetylasecomprising contacting the NAD⁺-dependent deacetylase with aNAD⁺-dependent deacetylase inhibiting amount of a compound of Formula I:

In Formula I, the letter X is a member selected from the groupconsisting of O and S. The symbols L¹ and L² each represent membersindependently selected from the group consisting of O, S, ethylene andpropylene, substituted with 0-2 R groups, wherein exactly one of thesymbols L¹ and L² represents a member selected from the group consistingof O and S. Each instance of the letter R of symbols L¹ and L²independently represents a member selected from the group consisting ofC₁₋₆alkyl, C₂₋₆alkenyl and —CO₂R⁴. The symbols R¹ and R² each representmembers independently selected from the group consisting of hydrogen,C₁₋₆alkoxy, C₀₋₆alkoxy-aryl and hydroxy. Alternatively, the symbols R¹and R² are taken together with the carbons to which they are attached toform a six-membered lactone ring. The symbol R³ represents a memberselected from the group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR⁴,—NR⁴R⁴, —CO₂R⁴, —C(O)R⁴, —C(O)NR⁴R⁴, —CN, —NO₂ and halogen. Eachinstance of the symbol R⁴ independently represents a member selectedfrom the group consisting of hydrogen and C₁₋₆alkyl.

In a preferred aspect of the present invention, the compound of FormulaI has the following structure:

In this case, the symbol R¹ is a member selected from the groupconsisting of hydrogen, C₁₋₆alkoxy and C₀₋₆alkoxy-aryl; the symbol R² isa member selected from the group consisting of hydrogen and hydroxy; thesymbol R³ is a member selected from the group consisting of hydrogen and—OR⁴; and the symbol R⁴ is C₁₋₆alkyl.

In another preferred aspect of the present invention, the symbol R¹ is amember selected from the group consisting of C₁₋₆alkoxy, C₀₋₆alkoxy-aryland hydroxy. In a more preferred aspect of the present invention, thesymbol R¹ is a member selected from the group consisting of hydroxy,methoxy and benzyloxy. In a most preferred aspect of the presentinvention, the symbol R¹ is benzyloxy. In another preferred embodiment,the term aryl is a member selected from the group consisting of phenyland naphthyl.

In another aspect of the present invention, a method is provided forinhibiting the deacetylase activity of a NAD⁺-dependent deacetylasecomprising contacting the NAD⁺-dependent deacetylase with aNAD⁺-dependent deacetylase inhibiting amount of a compound of FormulaII:

In Formula II, the symbol R^(a) is a member selected from the groupconsisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e), —NR^(e)R^(e),—CO₂R^(e), —C(O)R^(e), —C(O)NR^(e)R^(e), —CN, —NO₂ and halogen, whilethe symbol R^(b) is a member selected from the group consisting of:

In the components above, the symbol X^(a) represents a member selectedfrom the group consisting of O, S and NR^(e), while the symbol R^(c)represents a member selected from the group consisting of hydrogen,C₁₋₆alkyl and aryl optionally substituted with a member selected fromthe group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e),—NR^(e)R^(e), —CN, —NO₂ and halogen. The symbol R^(d) represents amember selected from the group consisting of hydrogen, C₁₋₆alkyl, aryl,—OR^(e), —NR^(e)R^(e) and halogen. And, each instance of the symbolR^(e) independently represents a member selected from the groupconsisting of hydrogen and C₁₋₆alkyl.

In a preferred aspect of the present invention, Formula II has thefollowing structure:

In a further aspect of the present invention, a method is provided forthe treatment of cancer comprising administering to a subject in need ofsuch treatment a first amount of an antineoplastic agent and a secondamount of a compound of Formula I:

In Formula I, the letter X is a member selected from the groupconsisting of O and S. The symbols L¹ and L² each represent membersindependently selected from the group consisting of O, S, ethylene andpropylene, substituted with 0-2 R groups, wherein exactly one of thesymbols L¹ and L² represents a member selected from the group consistingof O and S. Each instance of the letter R of symbols L¹ and L²independently represents a member selected from the group consisting ofC₁₋₆alkyl, C₂₋₆alkenyl and —CO₂R⁴. The symbols R¹ and R² each representmembers independently selected from the group consisting of hydrogen,C₁₋₆alkoxy, C₀₋₆alkoxy-aryl and hydroxy. Alternatively, the symbols R¹and R² are taken together with the carbons to which they are attached toform a six-membered lactone ring. The symbol R³ represents a memberselected from the group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR⁴,—NR⁴R⁴, —CO₂R⁴, —C(O)R⁴, —C(O)NR⁴R⁴, —CN, —NO₂ and halogen. Eachinstance of the symbol R⁴ independently represents a member selectedfrom the group consisting of hydrogen and C₁₋₆alkyl.

In another aspect of the present invention, a method is provided for thetreatment of cancer comprising administering to a subject in need ofsuch treatment a first amount of an antineoplastic agent, and a secondamount of a compound of Formula II:

In Formula II, the symbol R^(a) is a member selected from the groupconsisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e), —NR^(e)R^(e),—CO₂R^(e), —C(O)R^(e), —C(O)NR^(e)R^(e), —CN, —NO₂ and halogen, whilethe symbol R^(b) is a member selected from the group consisting of:

In the components above, the symbol X^(a) represents a member selectedfrom the group consisting of O, S and NR^(e), while the symbol R^(c)represents a member selected from the group consisting of hydrogen,C₁₋₆alkyl and aryl optionally substituted with a member selected fromthe group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e),—NR^(e)R^(e), —CN, —NO₂ and halogen. The symbol R^(d) represents amember selected from the group consisting of hydrogen, C₁₋₆alkyl, aryl,—OR^(e), —NR^(e)R^(e) and halogen. And, each instance of the symbolR^(e) independently represents a member selected from the groupconsisting of hydrogen and C₁₋₆alkyl.Compositions

In yet another aspect of the present invention, a composition isprovided for the treatment of cancer comprising an antineoplastic agentand a compound of Formula I:

In Formula I, the letter X is a member selected from the groupconsisting of O and S. The symbols L¹ and L² each represent membersindependently selected from the group consisting of O, S, ethylene andpropylene, substituted with 0-2 R groups, wherein exactly one of thesymbols L¹ and L² represents a member selected from the group consistingof O and S. Each instance of the letter R of symbols L¹ and L²independently represents a member selected from the group consisting ofC₁₋₆alkyl, C₂₋₆alkenyl and —CO₂R⁴. The symbols R¹ and R² each representmembers independently selected from the group consisting of hydrogen,C₁₋₆alkoxy, C₀₋₆alkoxy-aryl and hydroxy. Alternatively, the symbols R¹and R² are taken together with the carbons to which they are attached toform a six-membered lactone ring. The symbol R³ represents a memberselected from the group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR⁴,—NR⁴R⁴, —CO₂R⁴, —C(O)R⁴, —C(O)NR⁴R⁴, —CN, —NO₂ and halogen. Eachinstance of the symbol R⁴ independently represents a member selectedfrom the group consisting of hydrogen and C₁₋₆alkyl.

In a further aspect of the present invention, a composition is providedfor the treatment of cancer comprising an antineoplastic agent and acompound of Formula II:

In Formula II, the symbol R^(a) is a member selected from the groupconsisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e), —NR^(e)R^(e),—CO₂R^(e), —C(O)R^(e), —C(O)NR^(e)R^(e), —CN, —NO₂ and halogen, whilethe symbol R^(b) is a member selected from the group consisting of:

In the components above, the symbol X^(a) represents a member selectedfrom the group consisting of O, S and NR^(e), while the symbol R^(c)represents a member selected from the group consisting of hydrogen,C₁₋₆alkyl and aryl optionally substituted with a member selected fromthe group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e),—NR^(e)R^(e), —CN, —NO₂ and halogen. The symbol R^(d) represents amember selected from the group consisting of hydrogen, C₁₋₆alkyl, aryl,—OR^(e), —NR^(e)R^(e) and halogen. And, each instance of the symbolR^(e) independently represents a member selected from the groupconsisting of hydrogen and C₁₋₆alkyl.

In a preferred aspect of the present invention, the antineoplastic agentis a member selected from the group consisting of antiangiogenic andantivascular agents, antimetabolites, antifolates and other inhibitorsof DNA synthesis, antisense oligonucleotides, biological responsemodifiers, DNA-alkylating agents, DNA intercalators, DNA repair agents,growth factor receptor kinase inhibitors, hormone agents,immunoconjugates, microtubule disruptors and topoisomerase I/IIinhibitors.

In another preferred aspect of the present invention, the antineoplasticagent is a member selected from the group consisting ofcyclophosphamide, triethylenephosphoramide,triethylenethiophosphoramide, flutamide, altretamine,triethylenemelamine, trimethylolmelamine, meturedepa, uredepa,aminoglutethimide, L-asparaginase, BCNU, benzodepa, bleomycin, busulfan,camptothecin, capecitabine, carboquone, chlorambucil, cytarabine,dactinomycin, daunomycin, daunorubicin, docetaxol, doxorubicin,epirubicin, estramustine, dacarbazine, etoposide, fluorouracil,gemcitabine, hydroxyurea, ifosfamide, improsulfan, mercaptopurine,methotrexate, mitomycin, mitotane, mitoxantrone, novembrichin,paclitaxel, piposulfan, plicamycin, prednimustine, procarbazine,tamoxifen, temozolomide, teniposide, thioguanine, thiotepa, UFT, uracilmustard, vinblastine, vincristine, vinorelbine and vindesine.

In a further preferred aspect of the present invention, theantineoplastic agent is administered after the compound. In anotherpreferred aspect, the antineoplastic agent is administeredsimultaneously with the compound. In yet another preferred aspect, theantineoplastic agent is administered prior to the compound.

In another aspect of the present invention, a pharmaceutical compositionis provided, comprising a pharmaceutically acceptable excipient and acompound, and all pharmaceutically acceptable salts thereof, of FormulaI:

In Formula I, the letter X is a member selected from the groupconsisting of O and S. The symbols L¹ and L² each represent membersindependently selected from the group consisting of O, S, ethylene andpropylene, substituted with 0-2 R groups, wherein exactly one of thesymbols L¹ and L² represents a member selected from the group consistingof O and S. Each instance of the letter R of symbols L¹ and L²independently represents a member selected from the group consisting ofC₁₋₆alkyl, C₂₋₆alkenyl and —CO₂R⁴. The symbols R¹ and R² each representmembers independently selected from the group consisting of hydrogen,C₁₋₆alkoxy, C₀₋₆alkoxy-aryl and hydroxy. Alternatively, the symbols R¹and R² are taken together with the carbons to which they are attached toform a six-membered lactone ring. The symbol R³ represents a memberselected from the group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR⁴,—NR⁴R⁴, —CO₂R⁴, —C(O)R⁴, —C(O)NR⁴R⁴, —CN, —NO₂ and halogen. Eachinstance of the symbol R⁴ independently represents a member selectedfrom the group consisting of hydrogen and C₁₋₆alkyl.

In a preferred aspect of the present invention, the compound of FormulaI has the following structure:

In this case, the symbol R¹ is a member selected from the groupconsisting of hydrogen, C₁₋₆alkoxy and C₀₋₆alkoxy-aryl; the symbol R² isa member selected from the group consisting of hydrogen and hydroxy; thesymbol R³ is a member selected from the group consisting of hydrogen and—OR⁴; and the symbol R⁴ is C₁₋₆alkyl.

In another preferred aspect of the present invention, the symbol R¹ is amember selected from the group consisting of C₁₋₆alkoxy, C₀₋₆alkoxy-aryland hydroxy. In a more preferred aspect of the present invention, thesymbol R¹ is a member selected from the group consisting of hydroxy,methoxy and benzyloxy. In a most preferred aspect of the presentinvention, the symbol R¹ is benzyloxy. In another preferred embodiment,the term aryl is a member selected from the group consisting of phenyland naphthyl.

In another aspect of the present invention, a pharmaceutical compositionis provided, comprising a pharmaceutically acceptable excipient and acompound, and all pharmaceutically acceptable salts thereof, of FormulaII:

In Formula II, the symbol R^(a) is a member selected from the groupconsisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e), —NR^(e)R^(e),—CO₂R^(e), —C(O)R^(e), —C(O)NR^(e)R^(e), —CN, —NO₂ and halogen, whilethe symbol R^(b) is a member selected from the group consisting of:

In the components above, the symbol X^(a) represents a member selectedfrom the group consisting of O, S and NR^(e), while the symbol R^(c)represents a member selected from the group consisting of hydrogen,C₁₋₆alkyl and aryl optionally substituted with a member selected fromthe group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e),—NR^(e)R^(e), —CN, —NO₂ and halogen. The symbol R^(d) represents amember selected from the group consisting of hydrogen, C₁₋₆alkyl, aryl,—OR^(e), —NR^(e)R^(e) and halogen. And, each instance of the symbolR^(e) independently represents a member selected from the groupconsisting of hydrogen and C₁₋₆alkyl.

In a preferred aspect of the present invention, Formula II has thefollowing structure:

Administration

An effective amount of the composition will be determined by theexistence, nature, and extent of any adverse side-effects that accompanythe administration of the composition; the LD50 of the composition; andthe side-effects of the composition at various concentrations.Typically, the amount of the composition administered will range fromabout 0.01 to about 20 mg per kg, more typically about 0.05 to about 15mg per kg, even more typically about 0.1 to about 10 mg per kg bodyweight.

The compositions can be administered, for example, by intravenousinfusion, orally, intraperitoneally, or subcutaneously. Oraladministration is the preferred method of administration. Theformulations of compounds can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials.

The compositions of the present invention are typically formulated witha pharmaceutically acceptable carrier before administration to anindividual or subject. Pharmaceutically acceptable carriers aredetermined, in part, by the particular composition being administered,as well as by the particular method used to administer the composition.Accordingly, there are a wide variety of suitable formulations ofpharmaceutical compositions of the present invention (see, e.g.,Remington's Pharmaceutical Sciences, 17th ed., 1989).

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound of Formula I orFormula II, suspended in diluents, such as water, saline or PEG 400; (b)capsules, sachets or tablets, each containing a predetermined amount ofthe active ingredient, as liquids, solids, granules or gelatin; (c)suspensions in an appropriate liquid; and (d) suitable emulsions. Tabletforms can include one or more of the following: lactose, sucrose,mannitol, sorbitol, calcium phosphates, corn starch, potato starch,microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc,magnesium stearate, stearic acid, and other excipients, colorants,fillers, binders, diluents, buffering agents, moistening agents,preservatives, flavoring agents, dyes, disintegrating agents, andpharmaceutically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, e.g., sucrose, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin or sucrose and acacia emulsions, gels, and the like containing,in addition to the active ingredient, carriers known in the art.

The compositions of the present invention may be in formulationssuitable for other routes of administration, such as, for example,intravenous infusion, intraperitoneally, or subcutaneously. Theformulations include, for example, aqueous and non-aqueous, isotonicsterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. Injection solutions andsuspensions can be prepared from sterile powders, granules, and tablets.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. For example, if the compositions ofthe present invention are administered to treat or prevent cancer, suchas a tumor, the dose administered to the patient should be sufficient toprevent, retard, or reverse tumor growth. The dose will be determined bythe efficacy of the particular composition employed and the condition ofthe patient, as well as the body weight or surface area of the patientto be treated. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side-effects that accompanythe administration of a particular composition in a particular patient.

EXAMPLES

Compounds

The compounds of the present invention can be synthesized by severalmethods known to one of skill in the art. Methods for preparing the S1and S2 scaffolds are shown below by way of example, and are by no meanscomprehensive of the methods that can be used to synthesize thecompounds of the present invention. One of skill in the art willappreciate that the starting material, the reagants and the reactionsshown in the schemes below, can be appropriately modified in order tosynthesize all the compounds of the present invention. The appropriatemodifications are known to those of skill in the art.

Yeast Media

All strains can be grown in synthetic complete media (SC) or selectivesynthetic drop-out media containing 2% glucose.

Cell-Based Chemical Screen for the Sir2p Inhibitors: Inhibition ofSilencing Assays.

In order to find inhibitors of the deacetylase activity of Sir2p,screening was carried out to identify compounds that perturbed silencingat each of the loci at which Sir2p is known to act in S. cerevisiae:telomeres, HML, HMR, and the rDNA. The cell-based positive selectionscreen was designed so that inhibition of Sir2p activity permittednormal cell growth in order to avoid identifying cytotoxic compounds.Briefly, a yeast strain containing a marker gene, such as a nutritionalmarker, in close proximity to a telomeres in S. cerevisiae such that itis repressed by telomeric chromatin, is exposed to a test compound orset of test compounds dissolved in DMSO and cultured under suitableconditions and in media supplemented to permit growth only underconditions in which the marker gene is expressed. After a suitableinterval, the optical density of the culture is measured. An increase inoptical density corresponds to growth indicating a perturbation ofsilencing of the marker gene. All strains in this and other examplesherein can be grown in synthetic complete media (SC) or selectivesynthetic drop-out media containing 2% glucose.

Initial screening for compounds that effect silencing was carried outusing a URA3 assay. When the URA3 gene is in close proximity to atelomere in S. cerevisiae, it is repressed by telomeric chromatin(Gottschling, D. E., et al. (1990) Cell 63, 751-62). Because Ura3p isrequired for uracil biosynthesis, cells with the silenced telomeric URA3gene are unable to grow in media lacking uracil. Accordingly, geneticperturbation of silencing activates URA3 expression and enables cells togrow in the absence of uracil (Singer, M. S., et al., (1998) Genetics150, 613-3). Briefly, drug screening was performed in 96-well plates.Each well is inoculated with 150 μL of yeast culture (strain: UCC2210MATα ppr1 adh4::URA3::TEL(VII-L)), containing 1×10⁵ cell/ml inuracil-deficient media. A library of 6000 compounds from the NCIrepository was screened for those that disrupted telomeric silencing.The compounds dissolved in DMSO are applied at three differentconcentrations: 0.5, 5 and 50 μM. Cultures are incubated for 36-48 h andgrowth in individual wells is tested by optical density (OD₆₆₀)measurements and visual inspection. Eleven compounds identified in thisprimary screen were analyzed further to determine whether silencing atthe HML and HMR loci was also affected.

A secondary screening of the eleven identified compounds was carried outusing a TRP1 gene which utilized a yeast strain with a TRP1 geneintegrated at the silent HMR locus cannot grow in media lackingtryptophan (Buck, S. W. & Shore, D. (1995) Genes Dev. 9, 370-84). Usingthe method essentially described above, wells in a 96-well plate wereinoculated with 150 μL of yeast culture (a yeast strain with a TRP1 geneintegrated at the silent HMR locus), containing 1×10⁵ cell/ml intryptophan-deficient media. The eleven compounds dissolved in DMSO wereadded to the cells and the cultures are incubated for 36-48 h and growthin individual wells is tested by optical density (OD₆₆₀) measurementsand visual inspection. In this assay, one of the eleven compoundsenabled cells to grow in media lacking tryptophan (FIG. 1B), indicatingloss of silencing at HMR. This compound(1,2-dihydro-3H-naphtho[2,1-b]pyran-3-one, FIG. 1A), hereafter referredto as S1, also disrupted silencing at HML.

Effect of S1 on silencing at the HMLα locus in MATa cells.

In one assay to confirm that S1 was capable of inhibiting silencing invivo, a pheromone response assay was carried out. When haploid MATacells are exposed to the mating pheromone a factor, they arrest in G1phase of the cell cycle. Loss of silencing at the HMLα locus in MATacells results in expression of a mating type genes (Marsh, L., et al.(1991) Annu. Rev. Cell Biol. 7, 699-728). The coexpression of α and agenes creates a pseudo-diploid state: cells are immune to a factor andunable to mate. In the presence of S1, MATa cells lost responsiveness toα factor (FIG. 1C) and were defective for mating. Thus, treatment withS1 disrupted silencing at HML, HMR, and telomeric loci.

Effect of S1 on Recombination at the rDNA locus.

Sir2p is involved in the silencing of rDNA through a protein complexknown as RENT (regulator of nuclear silencing and telophase exit), whichdoes not include Sir3p or Sir4p and acts at the ribosomal RNA genecluster (rDNA). Silencing within the rDNA locus is manifested in twoways. It can weakly repress expression of an inserted reporter gene(Smith, J. S. & Boeke, J. D. (1997) Genes Dev. 11, 241-54), and itreduces recombination between tandem copies of the ribosomal RNA genes(Gottlieb, S. & Esposito, R. E. (1989) Cell 56, 771-6). Recombinationwas analyzed by measuring the loss rate of an ADE2 gene integrated intothe rDNA array essentially as described by Kaeberlein, M., et al. (1999Genes Dev. 13, 2570-80). A logarithmic culture of a yeast straincontaining an ADE2 gene integrated into the rDNA array was exposed to 15μM of S1 or DMSO for six hours. After six hours, the cultures wereplated onto rich medium and the loss of expression of the ADE2 gene wasmeasured and scored by the development of sectored red colonies. Theresults showed that S1 disrupted silencing of a reporter gene within therDNA locus, just as it did at telomeres and the HM loci. Treatment withSI increased recombination rate at the rDNA locus seven-fold, which issimilar to rates observed in a sir2 mutant (FIG. 1D). There was noeffect on rDNA recombination in sir2 cells treated with the compound,indicating that S1 was acting specifically through the SIR2 pathway.

Whole Gene Array Analysis For Transcriptional Profiling.

In addition to SIR2, the S. cerevisiae genome encodes four SIR2homologues: HST1-4 (Homologue of Sir Two) (Brachmann, C. B., et al.(1995) Genes Dev. 9, 2888-902). Hst2p is located in the cytoplasm and isresponsible for virtually all the NAD⁺-dependent deacetylase activitydetected in a cellular lysate (Smith, J. S., et al., (2000) Proc. Natl.Acad. Sci. USA 97, 6658-63). Its relevant biological substrate isunknown. Hst1p is required for transcriptional repression of meioticgenes (Xie, J., et al. (1999) EMBO J. 18, 6448-54), whereas little isknown about the cellular function of Hst3p or Hst4p. In order todetermine whether the anti-silencing effects of S1 were mediated solelyby Sir2p, and whether S1 affected any of the Hst proteins, theexpression profile of wild type cells grown in the presence of S1 wascompared to that of sir2, hst1, hst2, hst3 or hst4 deletion mutants bywhole genome DNA microarray analysis.

Strains for the DNA array experiments for the whole genome analysis wereobtained from Research Genetics (wild type BY4741: MATa his3, leu2,met15, ura4 or isogenic sir2, hst1, hst2, hst3 and hst4 deletionmutants). Several colonies from fresh cultures were inoculated intosynthetic complete medium with 2% glucose, grown overnight at 30° C.,diluted to 0.5−1×10⁶ cell/ml and grown for an additional 6-9 hours untilreaching a density of 0.5−1×10⁷ cells/ml. For experiments with S1, drugor the solvent (DMSO) was added at the beginning of the final 9-hourgrowth phase. In experiments with cycloheximide, cells were treated with50 μg/ml of cycloheximide for 40 minutes prior to the addition of S1.Total RNA was extracted using the hot acid phenol method. Microarrayconstruction and hybridization protocols were modified from thosedescribed elsewhere (DeRisi, J. L. et al. (1997) Science 278, 680-6).Briefly, yeast microarrays were constructed employing a set of ˜6200 orspecific PCR primer pairs (Research Genetics, Huntsville, Ala.), whichwere used to amplify each open reading frame (orf) of the yeast genome.Individual PCR products were verified as unique via gel electrophoresisand purified using ARRAYIT 96-well PCR purification kits (TeleChemInternational, Sunnyvale, Calif.). Purified PCR products weremechanically “spotted” in 3×SSC (450 mM sodium chloride and 45 mM sodiumcitrate, pH 7.0) onto poly-lysine coated microscope slides using anOMNIGRID high-precision robotic gridder (GeneMachines, San Carlo,Calif.).

The protocol used for cDNA labeling is a modification of a protocoldescribed elsewhere Briefly, labeled cDNA targets were prepared byreverse transcription of 30 .mu.g total RNA using oligo dT(18) primer inthe presence of 0.2 mM 5-(3-aminoallyl)-2′-deoxyuridine-5′-triphosphate(aa-dUTP; Sigma-Aldrich Company, St. Louis, Mo.), 0.3 mM dTTP, and 0.5mM each of dATP, dCTP, and dGTP. Following cDNA synthesis, either Cy3 orCy5 mono-reactive fluors (Amersham Life Sciences, Arlington Heights,Ill.) were covalently coupled to the cDNA-incorporated aminoallyl linkerin the presence of 50 mM sodium bicarbonate (pH 9.0). Two colorexpression profiles were generated using microarrays in which referenceand experimental cDNA targets were labeled with different fluors.Following co-hybridization to the chip, a fluorescent image of themicroarray was collected at both emission wavelengths using a GenePix4000 fluorescent scanner (Axon Instruments, Inc., Foster City, Calif.)and image analysis is performed using GenePix Pro Microarray Acquisitionand Analysis Software.

Three competitive hybridizations for each experimental group (sir2,hst1, hst2, hst3 and hst4 versus wild type, S1 treated wild type versuswild type and S1 plus cycloheximide versus cycloheximide alone) wereperformed using three separate cultures and log₂ of the expression ratiocalculated for every ORF. To assess the intrinsic variation ofexpression level for different ORF's, nine wild type versus wild typehybridizations were performed using nine separate cultures. Studentt-test was used to assess if the difference between the log₂ of theexpression ratio for ORF in the experimental and control group (wildtype versus wild type) was significant.

Using this array method, the transcriptional effects of S1 correlatedmost highly with those of a sir2 mutation (correlation coefficient0.748, FIG. 2A). Genes adjacent to telomeres such as COS12, and the α1and α2 genes from the HML locus, were significantly up-regulated in bothconditions (FIG. 2A). The expression of MATa-specific (e.g. MFA1, STE2,STE6, BAR1) and haploid-specific genes (e.g. FUS1, STE5) wasdown-regulated in both S1 treated cells and in sir2 cells (FIGS. 2, Aand B). S1 also up-regulated a small number of genes that were notaltered in sir2 cells, including meiosis specific genes (e.g. SPS1)which appear to be regulated by HST1 (FIG. 2B). There was no overlapbetween S1 and HST2, HST3, or HST4 regulated genes (FIG. 2D). Thus themajority of all transcriptional changes (88%) induced by S1 weremediated through SIR2 and a smaller subset (9%) through HST1 (FIG. 2B).These results indicate that S1 is a selective Sir2p inhibitor.

Sir2p is critical for silencing, yet the majority of the transcriptionalchanges induced by either chemical or genetic inactivation of the enzymeconstituted transcriptional down-regulation (FIGS. 2, A and B). A numberof these changes are known to be indirect. For instance, haploidspecific genes are down-regulated by the gene products of thederepressed HMLα and HMRa loci (Marsh, L., et al. (1991) Annu. Rev. CellBiol. 7, 699-728). S1, combined with the protein synthesis inhibitorcycloheximide, afforded an opportunity to identify genes that aredirectly regulated by Sir2p. Such an examination has not been possiblebefore because conditional alleles of SIR2 are not available. Theaddition of cycloheximide did not affect the upregulation of genes by S1treatment. In contrast, virtually all transcriptional down-regulationwas abolished in the absence of new protein synthesis. This confirmedthat the direct effect of Sir2p is to repress transcription (FIG. 2C).With the exception of a single gene, BPH1, the only genes that wereup-regulated as a result of Sir2p inactivation in the presence ofcycloheximide were subtelomeric genes and silent mating type loci,indicating that Sir2p activity does not affect transcription outside ofthese regions. Overall, these results are consistent with a recent studyexamining the location of Sir2p by genome-wide chromatinimmunoprecipitation (Lieb, J. D., et al. (2001) Nat. Genet. 28, 327-34).

HDA Assay for Determination of Inhibition of Deacetylase Activity ofSir2p by S1.

Without being bound to any particular theory, the phenotypic changescaused by S1 are thought to be the result of inhibition of the histonedeacetylase activity of Sir2p. Accordingly, S1 was evaluated for itsability to inhibit the histone deacetylase activity of Sir2p in vitro.An [³H]-acetylated histone H4 peptide was used in the assay whichmeasured the NAD⁺-dependent release of free [³H]-acetate in the presenceof whole yeast cell extract from an hst2 strain overexpressing yeastSIR2. A cell extract obtained from a SIR2 overexpressing hst2 strain hadrobust NAD⁺-dependent histone deacetylase activity derived exclusivelyfrom Sir2p (FIGS. 3, A and B).

Briefly, histone H4 was chemically acetylated using the HDAC Assay Kit(Upstate Biotechnology). The whole cell extract is prepared as described(Smith, J. S. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 6658-63) froman hst2Δ strain containing 2 μ plasmid with galactose-inducible wildtype SIR2 (pAR14, Braunstein, M. et al. (1993) Genes Dev. 7, 592-604) ormutant SIR2 (GAL-SIR2-Y298N or GAL-SIR2-H286Q) or empty vector. For thehistone deacetylase assays, 50 μg of yeast whole-cell protein extractwas incubated with [³H] acetylated histone H4-peptide (40,000 cpm) withor without 500 μM NAD⁺ in a 100 μl reaction. The buffer contained 150 mMNaCl, 25 mM sodium phosphate pH 7.4 and 1 mM DTT. Reactions wereincubated at 30° C. for 16 hours and were stopped by the addition of 25μl of 1 N HCl and 0.15 N acetic acid. Released [³H] acetate wasextracted with 400 μl of ethyl acetate. S1 induced dose dependentinhibition of histone deacetylase activity in the yeast extract, with anIC50 of 60 μM (FIG. 3A). This result established Sir2p deacetylaseactivity as a direct target of S1.

Preparation and Identification of Mutations Conferring Drug Resistanceto S1.

To obtain further insight into the molecular mechanism by which S Iinhibited deacetylase activity of Sir2p, mutant forms of Sir2p weregenerated that were resistant to the compound. The conserved core regionof SIR2 was amplified using error prone PCR and was integrated into aSIR2 containing centromeric plasmid (pRS314-SIR2) by cotransformationinto a sir2Δ strain with a URA3 telomeric marker (strain AB14053 MATαsir2 ppr1 adh4::URA3::TEL(VII-L)). Transformants from selective (-trp)media are pooled and aliquots plated onto selective media containing5-fluoroorotic acid (5-FOA) and 10 μM S1. Plasmid DNA was recovered fromthe individual colonies and was retransformed into the test strain toassure that drug resistance was conferred by SIR2-containing plasmid.The entire SIR2 open reading frames from 20 independent plasmidsconferring S1 resistance were sequenced. Mutations were introduced intoa plasmid containing galactose inducible SIR2 (pAR14 (Braunstein, M. etal. (1993) Genes Dev. 7, 592-604)) using gap repair or site directedmutagenesis to make GAL-SIR2-Y298Nand GAL-SIR2-H286Q. Three alleles ofSIR2 (SIR2-H286Q, SIR2-L287Q and SIR2-Y298N)were identified that renderyeast cells resistant to the anti-silencing effects of S1. Silencing wasat normal levels in the drug resistant mutants in the absence of drug,but disruption of silencing in the mutants required higherconcentrations of S1 than in wild type strains (FIG. 3C). In vitro, whencompared to equivalent amounts of wild type Sir2p, mutant proteinsexhibited similar histone deacetylase activity in the absence of drug,with increased resistance to the inhibitory effect of S1 (FIGS. 3, A andB).

The three mutations lie in close proximity within a region that ishighly similar to human SIRT2. Most interestingly, the crystal structureof SIRT2 defines this region to be a hydrophobic cavity that ishypothesized to be the binding site for acetylated lysine peptides(Finnin, M. S., et al. (2001) Nat. Struct. Biol. 8, 621-5 and Min, J.,et al. (2001) Cell. 105, 269-79.). As noted above, the expressionprofile of S1-treated cells had no overlap with mutant hst2, hst3, orhst4 strains, but did have some overlap with the hst1 profile. Of allthe HST genes, Hst1p has the highest sequence similarity (86% identity)to Sir2p in the 50 amino acid region containing the S1 resistancemutations (FIG. 3D). Since Hst1p also acts to repress gene expressionvia hypoacetylation of histones (Rusche, L. N. & Rine, J. (2001) GenesDev. 15, 955-67 and Sutton, A., et al. (2001) Mol. Cell Biol. 21,3514-22.), it seems likely that this shared region defines a commonbinding pocket for acetylated histone tails in both proteins. Thus, itappears that S1 inhibits the deacetylase activity of Sir2p by blockingaccess to the acetylated histone binding pocket, or by altering theconfirmation of the acetylated histone binding pocket, such that thedeacetylase activity of Sir2p is inhibited.

Continuous Deacetylase Activity of Sir2p is Required for the Maintenanceof the Silent State in Non-Dividing Cells.

The establishment of silencing in previously active chromatin is a cellcycle dependent event that can be accomplished only during S-phase (Li,Y. C., et al. (2001) Science 291, 650-3 and Kirchmaier, A. L. & Rine, J.(2001) Science 291, 646-50). Once established, the silent state needs tobe maintained between cell divisions, after mitosis, in G1 and into thenext S phase. Studies with a temperature sensitive allele of SIR3demonstrated that silencing is quickly lost in G1-arrested cells aftercells are shifted to the nonpermissive temperature (Miller, A. M. &Nasmyth, K. A. (1984) Nature 312, 247-51). In contrast, removal of theDNA silencer elements from the HMLα locus in G1-arrested cells does notdisrupt silencing (Holmes, S. G. & Broach, J. R. (1996) Genes Dev. 10,1021-32). The study with the temperature sensitive allele of SIR3suggests that the presence of the entire SIR complex is required for themaintenance of a silent state.

The requirement for the deacetylase activity of Sir2p for themaintenance of a silent state in non-dividing cells or whether Sir2p wasdispensable once silent chromatin was formed was not established. Toaddress this issue, the ability of S1 to inhibit the histone deacetylaseactivity of Sir2p was used. Briefly, MA Ta cells were first arrested inG1 using α factor and then treated with S1. While untreated cellsremained arrested in G1, those treated with S1 progressed through thecell cycle (FIG. 4A). This was presumably due to loss of matingcompetence, a consequence of expression of the α2 gene from the “silent”HML locus. To test this idea more directly, a MATa strain with a singleG1 cyclin gene (CLN3), which is under control of a galactose-induciblepromoter (Cross, F. R. (1990) Mol. Cell Biol. 10, 6482-90), was arrestedin G1 by replacing galactose with glucose in the media. Once the cellsarrested in G1, they were treated with S1 or a DMSO control. While thecells remained arrested in G1 under both conditions, α2 mRNA expressionfrom HML was detected only in the S1-treated cells (FIG. 4B). The lagperiod of several hours between the application of S1 and the appearanceof α2 mRNA was similar to the delay before cell cycle progression wasobserved in the a factor arrested cells treated with S1 (describedabove). These results demonstrated that the deacetylase activity ofSir2p is continuously required for the maintenance of the silent statein non-dividing cells.

As a result of these studies, Sir2p must remain diligent in maintainingthe silent state, in order to counteract the constant activity ofhistone acetylases. The acetylases may gain access to the chromatin in atargeted manner via transcriptional activators (Aparicio, O. M. &Gottschling, D. E. (1994) Genes Dev. 8, 1133-46 and Sekinger, E. A. &Gross, D. S. (2001) Cell 105, 403-14) or be part of a global histoneacetylation maintenance system (Vogelauer, M., et al. (2000) Nature 408,495-8). These results also support the idea that silent chromatin is nota static, rigid structure, but rather that it is in a dynamicequilibrium with silencing factor exchanging on and off the chromatin,even when cells are not dividing (Cheng, T. H. & Gartenberg, M. R.(2000) Genes Dev. 14, 452-63).

These results underscore the power of phenotypic screening in modelsystems to identify new compounds that are useful for dissecting complexbiological processes such as silencing in vivo. To this end, theidentification of an inhibitor of Sir2p complements the existinginhibitors of histone deacetylases (i.e. trapoxin and trichostatin(Taunton, J., et al. (1996) Science 272, 408-11)). In addition tohistones, many other proteins are regulated by acetylation, includingpRb, E2F and p53 proteins (Chan, H. M., et al. (2001) Nat. Cell Biol.3,667-74; Martinez-Balbas, M. A., et al. (2000) EMBO J. 19,662-71; andLuo, J., et al. (2000) Nature 408, 377-81). Two recent reports (Vaziri,H., et al. (2001) Cell 107, 149-159 and Luo, J., et al. (2001) Cell 107,137-148) implicate deacetylation of p53, by Sir2, in down-regulation oftranscriptional and proapoptotic activities of p53 in response to DNAdamage. Toxicity assays using S1 and a variety of DNA damaging agentshave shown that S1 sensitizes mammalian cells to these agents,consistent with S1 abrogating Sir2p activity on p53. Thus, S1 is auseful component in the evaluation of Sir2p-like deacetylases as drugtargets for treating cancer and other diseases (Wolffe, A. P. (2001)Oncogene 20, 2988-90 and Tycko, B. & Ashkenas, J. (2000) J. Clin. Invest105, 245-6).

S1 Sensitization of Mammalian Cells to DNA Damage.

To test whether S1 could sensitize cells to DNA damage, mammalian cellswere exposed to etoposide, a DNA damaging agent, in the presence andabsence of S1. Briefly, Rat1a and primary human fibroblasts (HFF) weretreated with etoposide alone (0) or with S1 25, 50 and 100 μM) andetoposide for 72 hours. Viability of cells was assessed with3H-thymidine incorporation. Viability of cells in etoposide relative toviability without etoposide is graphed for every concentration of S1. Inboth Rat1a cells and human foreskin fibroblast cells, S1 induced adose-dependent sensitization to etoposide (FIG. 5).

FIG. 6 is a table of compounds useful in the present invention, andtheir activity as determined in the assays described above.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. In addition,each reference provided herein is incorporated by reference in itsentirety to the same extent as if each reference was individuallyincorporated by reference.

1. A method for treating lymphoma, comprising the step of administeringto a subject in need thereof a therapeutically effective amount of acompound of Formula II:

wherein R^(a) is a member selected from the group consisting ofhydrogen, C₁₋₆alkyl, aryl, —OR^(e), —NR^(e)R^(e), —CO₂R^(e), —C(O)R^(e),—C(O)NR^(e)R^(e), —CN, —NO₂ and halogen; R^(b) is:

X^(a) is a member selected from the group consisting of O, S and NR^(e);R^(c) is a member selected from the group consisting of hydrogen,C₁₋₆alkyl and aryl optionally substituted with a member selected fromthe group consisting of hydrogen, C₁₋₆alkyl, aryl, —OR^(e),—NR^(e)R^(e), —CN, —NO₂ and halogen; and each instance of R^(e) isindependently a member selected from the group consisting of hydrogenand C₁₋₆alkyl.
 2. The method of claim 1, wherein said method is for thetreatment of a B-cell-derived non-Hodgkin lymphoma or a diffuse largeB-cell lymphoma.
 3. The method of claim 1, wherein said compound has theformula:


4. The method of claim 1, wherein said compound is administered with anantineoplastic agent.
 5. The method of claim 4, wherein saidantineoplastic agent is a member selected from the group consisting ofantimetabolites, antifolates and other inhibitors of DNA synthesis,antisense oligonucleotides, biological response modifiers,DNA-alkylating agents, DNA intercalators, DNA repair agents, growthfactor receptor kinase inhibitors, hormone agents, immunoconjugates,microtubule disruptors and topoisomerase I/II inhibitors.
 6. A method ofclaim 5, wherein said antineoplastic agent is a member selected from thegroup consisting of cyclophosphamide, triethylenephosphoramide,triethylenethiophosphoramide, BCNU, bleomycin, busulfan, chlorambucil,cytarabine, dactinomycin, daunorubicin, docetaxol, doxorubicin,epirubicin, estramustine, dacarbazine, etoposide, fluorouracil,gemcitabine, ifosfamide, methotrexate, mitomycin, mitoxantrone,paclitaxel, procarbazine, temozolomide, teniposide, thioguanine, uracilmustard, vinblastine, vincristine, vinorelbine and vindesine.
 7. Themethod of claim 6, wherein said antineoplastic agent is administeredafter said compound.
 8. The method of claim 6, wherein saidantineoplastic agent is administered simultaneously with said compound.9. The method of claim 6, wherein said antineoplastic agent isadministered prior to said compound.
 10. The method of claim 1, whereinsaid lymphoma has metastasized.